Age-Dependent Remodeling in Infrapatellar Fat Pad Adipocytes and Extracellular Matrix: A Comparative Study

Abstract

The infrapatellar fat pad (IFP) is actively involved in knee osteoarthritis (OA). However, a proper description of which developmental modifications occur in the IFP along with age and in absence of joint pathological conditions, is required to adequately describe its actual contribution in OA pathophysiology. Here, two IFP sources were compared: (a) IFP from healthy young patients undergoing anterior-cruciate ligament (ACL) reconstruction for ACL rupture (n = 24); (b) IFP from elderly cadaver donors (n = 23). After histopathological score assignment to confirm the absence of inflammatory features (i.e., inflammatory infiltrate and increased vascularity), the adipocytes morphology was determined; moreover, extracellular matrix proteins were studied through histology and Second Harmonic Generation approach, to determine collagens content and orientation by Fast Fourier Transform and OrientationJ. The two groups were matched for body mass index. No inflammatory signs were observed, while higher area, perimeter, and equivalent diameter and volume were detected for the adipocytes in the elderly group. Collagen III displayed higher values in the young group and a lower total collagen deposition with aging was identified. However, collagen I/III ratio and the global architecture of the samples were not affected. A higher content in elastic fibers was observed around the adipocytes for the ACL-IFPs and in the septa cadaver donor-IFPs, respectively. Age affects the characteristics of the IFP tissue also in absence of a pathological condition. Variable mechanical stimulation, depending on age-related different mobility, could be speculated to exert a role in tissue remodeling.

Keywords: aging; anterior-cruciate ligament rupture; cadaver donors; extracellular matrix; infrapatellar fat pad; osteoarthritis.

Figure 1

Inflammatory infiltration and vascularity. No lymphocytic infiltration was detected in both groups. H&E staining showing vessels distribution (black arrows) in ACL-IFP and cadaver donors-IFP (scale bars=100 μm). Vascularity is comparable between the two experimental groups. In both groups it is clearly recognizable the cross-section of the blood vessels lumen, lined by the endothelial cells.

Figure 2

Adipocytes morphometric analysis. (A) After H&E staining of ACL-IFPs (scale bar = 100 μm) and cadaver donor-IFPs (scale bar = 200 μm), the images were processed. The adipocytes were manually identified (see the red-line profile); thus, the images were converted into the corresponding 8-bit gray-level images for the measurements of mean adipocytes area (μm²). As showed in the graphs, a significant difference (p < 0.0001) was detected between the two experimental groups for the adipocytes area (B) but also for the adipocytes perimeter (C), equivalent diameter (D) and equivalent volume (E); ACL-IFPs samples n = 24 and cadaver donors-IFPs n = 23 samples. Data are reported as the median and interquartile range (IQR).

Figure 3

Collagen type I and III distribution. Sirius red stain under polarized light was performed to assess collagen type I (appearing red-orange) and type III (appearing green-yellow) distribution in ACL-IFPs (n = 18) (A) and cadaver donors-IFPs (n = 17) (scale bar = 50 μm). Collagens amount was then calculated in the two groups; any difference was observed in collagen type I content (B); conversely, collagen type III showed significantly higher values for the ACL-IFPs than for cadaver donors (p = 0.004) (C). The ratio between the collagens type I/III (Col I/Col III) was then highlighted; any significant difference was observed between the two groups (D). The data analysis considered n=18 tissue samples for ACL-IFPs and n = 16 tissue samples for cadaver donor derived-IFPs. Data are reported as the median and IQR. AU, arbitrary unit.

Figure 4

Evaluation of collagen content and distribution. (A) Representative images of ACL-IFP and cadaver donor-IFP samples after label-free Second Harmonic Generation (SHG) collagen analysis (scale bar = 50 μm). (B) SHG intensity evaluation: a significant difference (p = 0.0002) was detected for total collagen content in the cohort, with higher values in the ACL-IFP group than in the cadaver donor-IFP group. (C) Fast Fourier Transforms (FFTs) applied to ACL-IFP and cadaver donor-IFP samples showing a circular and an oval collagens orientation for ACL-IFPs and cadaver donor-IFPs, respectively. (D) FFT power plots in (C) were fitted with an ellipse and the ratio of long and short axis is reported. (E) Hue, Saturation and Brightness (HSB) maps obtained through the OrientationJ plugin for the ACL-IFP and cadaver donor-IFP samples; in these maps, to an orientation angle of the collagen is assigned a color and a specific color saturation (scale bar = 100 μm). (F) Coherency analysis did not show significant differences in the cohort.

Figure 5

Elastic fibers distribution. Van Gieson staining showed in violet the elastic fibers orientation around the adipocytes (A) (scale bar = 200 μm) and in correspondence of the septa (B) (scale bar = 50 μm) for ACL-IFPs and cadaver donors-IFPs. Quantification of the elastic fibers showed differences in the two settings. Significantly, higher elastic fibers were observed around the adipocytes in the ACL-IFPs (p = 0.021) (C) but not in correspondence of the septa in the cadaver donors-IFPs (p = 0.0872) (D). For the adipocytes surrounding areas, data analysis considered n = 12 and n = 23 tissue samples for ACL-IFPs and cadaver donors-IFPs, respectively; for the septa, data analysis considered n = 5 and n = 23 tissue samples for ACL-IFPs and cadaver donors-IFPs, respectively. AU, arbitrary unit.

Figure 6

Existing correlations in the overall patient's cohort. Collagen III and elastic fibers content were negatively correlated with age (A,C) and adipocytes area (B,D). Adipocytes area was positively correlated with age (E).

Figure 7

Comparative picture between the two study groups, showing the tissutal specific features related to IFP physiological maturation, in case of the absence of carilage defect. The image highlights the main study findings with respect to the initial hypotheses which have been identified in the red (a. absence of inflammatory features in case of no cartilage lesions), green (b. variation in adipocytes area despite manteinence of the typical cells morphology with aging) and blue (c. reduction in extracellular matrix proteins and different distribution with aging) boxes, respectively. ACL, anterior cruciate ligament reconstruction; BMI; body mass index; Col, collagen; EF, elastic fibers; IFP, infrapatellar fat pad.